Site Of Retention Research Articles

EFFECT OF HUMAN OVIDUCTAL EPITHELIAL CELL CULTURAL MEDIUM ON CRYOPRESERVED HUMAN SPERM SURVIVAL

The human oviduct is known as a functional site for gamete transportation, retention, fertilization and zygote development. Previous studies have shown that human oviductal epithelial cell cultural medium (OECCM) has a positive effect on prolongation of sperm motility for some cryopreserved human sperm without cryodamage. However, for most cryopreserved sperm, OECCM could not improve their survival prolongation. In this study, we assessed the influence of human OECCM on the motility longevity of cryopreserved human sperm with an in vitro incubation method.

Astrocytes as a predominant cellular site of (99m)Tc-HMPAO retention.

Technetium-99m-d,l-hexamethylpropylene amine oxime ((99m)Tc-HMPAO) retention in the brain monitored by single photon emission computed tomography (SPECT) is currently used as a marker of cerebral blood flow. The purported mechanism by which (99m)Tc-HMPAO accumulates in the brain is through its intracellular conversion from a lipophilic form to more hydrophilic derivatives within the brain parenchyma. The issue of the contribution of different cell types on (99m)Tc-HMPAO retention was investigated in vitro by studying the accumulation of (99m)Tc-HMPAO in primary cultures of mouse cortical astrocytes and neurons. Results show that (99m)Tc-HMPAO retention predominates in astrocytes over neurons by a factor of approximately 2.5 (0.26 +/- 0.05 vs. 0.095 +/- 0.042 fmol/mg protein after 120 minutes, respectively). Diethyl maleate (60 micromol/L), ethacrynic acid (1 mmol/L) and buthionine sulfoximine (1 mmol/L), 3 agents which significantly reduced glutathione levels also decreased (99m)Tc-HMPAO retention in both astrocytes (29%, 3%, and 46% of control, respectively) and neurons (69%, 11% and 63% of control). Decrease did not always correlate with glutathione levels, however, which suggests that other factors could be involved. The possibility that cell energy status determines (99m)Tc-HMPAO retention was also assessed. Agents that activate (glutamate, azide) or inhibit (cytochalasin B) glucose utilization in astrocytes, as measured by the (3)H-2-deoxyglucose method, were without effect on (99m)Tc-HMPAO retention. In conclusion, the data presented indicate that astrocytes may constitute a prominent site of (99m)Tc-HMPAO retention and most likely contribute significantly to the SPECT signal. In addition, the data also suggest that specific alterations in glial cell metabolism could explain flow-independent changes in (99m)Tc-HMPAO retention in the brain as observed by SPECT in some pathologies.

Collecting duct is a site of sodium retention in PAN nephrosis: a rationale for amiloride therapy.

Micropuncture studies of the distal nephron and measurements of Na,K-ATPase activity in microdissected collecting tubules have suggested that renal retention of sodium in puromycin aminonucleoside (PAN) nephrotic rats originates in the collecting duct. The present study demonstrated this hypothesis by in vitro microperfusion and showed that amiloride was able to restore sodium balance. Indeed, isolated perfused cortical collecting ducts from PAN-treated rats exhibited an abnormally high transepithelial sodium reabsorption that was abolished by amiloride, and in vivo administration of amiloride fully prevented decreased urinary sodium excretion and positive sodium balance in nephrotic rats. As expected from the aldosterone independence of Na(+) retention in PAN nephrotic rats, blockade of aldosterone receptor by potassium canrenoate did not alter urinary Na(+) excretion, Na(+) balance, or ascites formation in PAN nephrotic rats.

Human FDC express PrPc in vivo and in vitro.

Prion diseases are fatal neurodegenerative disorders caused by accumulation of abnormal prion protein (protease-resistant prion, PrPres). PrPres accumulation is also detected in lymphoid organs after peripheral infection. Several studies suggest that follicular dendritic cells (FDC) could be the site of PrPres retention and amplification. Here we show that human follicular dendritic cells can express normal cellular prion protein (PrPc) both in situ and in vitro. When tonsillar cryosections were treated with anti-PrP antibody, the label was found on some very delicate cell extensions inside the lymphoid follicles, especially in the germinal centres. These extensions react with DRC1 antibody, used frequently to label FDC. Other structures labelled with anti-PrP antibody were the keratinocytes. To confirm the ability of FDC to synthesise PrPc, we isolated FDC by a non-enzymatic procedure and cultured them. By cytochemistry and flow cytometry it was clearly shown that FDC do produce PrPc.

A Biokinetic Model for Alpha-emitting Bone Surface-seeking Radionuclides in the Mouse Skeleton

Following intake of an alpha-emitting bone surface-seeking radionuclide, such as 239Pu or 241Am, a major site of retention is the skeleton. Within the skeleton, these radionuclides are initially deposited on the bone surfaces, but subsequently redistributed as a result of the process of bone remodelling. In this study, these processes have been modelled in the mouse femoral shaft, by development of a multicompartmental biokinetic model. The model consists of bone surface, volume and marrow and blood compartments, each of which are described by input parameters. The majority of parameter values were obtained from current literature, and the remainder obtained by fitting of the model to microdistribution and radiation dosimetry data taken from previous autoradiographical analyses of the mouse femoral shaft, at times from 1 to 448 days after a 40 kBq.kg-1 injection of either 239Pu or 241Am. Results of fitting the model to these data predict that (a) 239Pu and 241Am show equal affinities for both resorbing and forming bone surfaces, (b) 241Am is retained in macrophages for a longer period than 239Pu and, (c) the cumulative dose to the region of marrow containing the osteosarcomagenic target cells is higher for 239Pu than 241Am by a factor of two.

The effect of brachium conjunctivum transection on a conditioned limb response in the cat

Seven cats were trained to perform a forelimb conditioned response to a paired tone conditioned stimulus (CS)/shock unconditioned stimulus (UCS). Brachium conjunctivum section ipsilateral to the trained limb was carried out following criterion conditioned response (CR) performance. Lesion sites were identified histologically and further confirmed by observation of cellular changes in the dentate and interpositus nuclei ipsilateral to the section. The brachium conjunctivum was found to have been sectioned in four of the seven subjects. Each of these animals demonstrated a total or near-total loss of the CR. Extended postoperative training resulted in no increase in CR performance levels. The unconditioned response (UCR) remained unaffected, as did limb placing, accuracy of striking at moving objects, grooming, running and walking. The results are discussed in the context of an earlier report by McCormick et al. [Bull Psychonom Soc 1981;18:103–5], in which section of the superior cerebellar peduncle was found to abolish a conditioned nictitating membrane response in the rabbit. Taken together, they support the contention of Lavond [Annu Rev Psychol 1993;44:317–42], Thompson [In: Sprague JM, Epstein AN, editors. Progress in Psychobiology and Physiological Psychology. New York: Academic Press 1983, pp. 167–96], Yeo et al. [Behav Brain Res 1984;13:261–66; Exp Brain Res 1985;60:87–98; Exp Brain Res 1985;60:99–113; Exp Brain Res 1992;88:623–38.] and others that the cerebellum represents a critical site for acquisition and retention of a conditioned memory trace.

Melanin affinity: a possible explanation of isoxsuprine retention in the horse

Isoxsuprine is used in veterinary medicine as a vasodilating agent. The drug has been detected in the urine of horses up to 6 weeks after the cessation of administration. In the present study, the distribution pattern of 3H-isoxsuprine was investigated using whole body autoradiography in mice to find a possible site of retention. Melanin was the only place of retention identified. Additional in vitro studies showed an affinity of isoxsuprine to both melanin and keratin. The K(d) values were 0.02 mmol/l and 1 mmol/l, and the B(max) values were 0.2 micromol/mg and 2 micromol/mg, respectively. A low affinity site with approximately the same K(d) and B(max) as keratin was also detected for melanin. 3H-isoxsuprine was found to have affinity to pigmented horse skin after incubation in vitro and microautoradiography. We believe that affinity to melanin and possibly also to keratin can cause retention of the drug in the body and therefore explain the prolonged excretion of low levels of isoxsuprine in the horse.

Anatomy and physiology of the gastrointestinal tract of the Julia Creek dunnart, Sminthopsis douglasi (Marsupialia : Dasyuridae)

The gastrointestinal tract of the endangered Julia Creek dunnart (Sminthopsis douglasi), the largest member of the genus Sminthopsis, consists of a simple, unilocular stomach and an intestine of relatively uniform calibre throughout. There is no hindgut caecum, in common with other Australian carnivorous marsupials. Brunner’s glands form a collar at the proximal end of the duodenum; they consist of simple uncoiled tubes at Day 45 of pouch life but are well differentiated at Day 60, before the young take their first solid food at Day 65–70. Rate of passage of digesta was measured in nine adult Julia Creek dunnarts on diets of minced meat with either mealworm larvae or adult crickets added, using pulse doses of the solute marker Co–EDTA and large (0.5–1.0 mm) particles of plant cell walls mordanted with Cr. Transit time (time of first appearance in the faeces) of both markers (P < 0.001) and mean retention time (the average time markers are retained in the tract) of the solute marker (P < 0.05) were shorter on the diet containing mealworms than the cricket diet. These results suggest that emptying of the stomach (the main site of digesta retention in carnivores) was delayed on the cricket diet, possibly because of longer digestion times as a result of a tougher exoskeleton. Comparison with other data suggests that total tract passage times increase among dasyurids as body size increases.

Role of the renal collecting system in initial kidney stone formation.

The anatomic site where the initial kidney stone grows to a clinically significant size is unknown. We studied human forniceal anatomy by microdissection and correlated the anatomic findings with the clinical presentation of kidney stones. In addition, we examined crystal attachment sites within the collecting system by direct infusion of calcium oxalate crystals into the renal pelvis of rabbits. Secondary urinary dead spaces were found in the fornices of compound papillae only, which were located at the poles of the kidneys. This feature correlated with a higher incidence of stones in these areas at the time of lithotripsy. Calcium oxalate crystals tended not to attach to either the renal papilla nor the pelvic sidewall unless these epithelia were previously injured with hydrochloric acid. The renal collecting system may serve as an anatomic site for crystal retention and growth but is unlikely to be the principal site for crystal attachment, unless prior cellular injury occurs.

Digesta retention in the gastro-intestinal tract of the orang utan (Pongo pygmaeus)

The diet of the orang utanPongo pygmaeus consists of fruit, leaves, communal insects, and bark, and contains appreciable amounts of non-starch polysaccharides. These complex carbohydrates require microbial fermentation before they can be used as an energy source by the orang utans. The gastrointestinal tract ofP. pygmaeus consists of a simple or unipartite stomach, a relatively long small intestine, and a complex haustrated caecum and colon. This morphology suggests that the capacious proximal colon is the principal site of digesta retention and fermentation of non-starch polysaccharides. We measured several parameters of digesta retention by giving three captive adultP. pygmaeus a pulse dose of inert markers specific for the solute and particulate phases of the digesta and collected their faeces at regular intervals over 192–338 hours. Transit times (times of first appearance of the markers in the faeces) and mean retention times (MRT) were long, consistent with a large complex gastro-intestinal tract. MRTs for the particulate marker were longer (p=0.032) than for the solute marker, indicative of selective retention of large particulate digesta. These results are consistent with the patterns of marker excretion in other mammals that use the digestive strategy of colon fermentation.

Glomerulonephritis and sodium retention: enhancement of Na+/K+-ATPase activity in the collecting duct is shared by rats with puromycin induced nephrotic syndrome and mice with spontaneous lupus-like glomerulonephritis.

In rats with puromycin aminoglucoside-induced (PAN) nephrotic syndrome, micropuncture studies have localized the site of sodium retention to the collecting duct. We have confirmed this finding by demonstrating a two-fold increase in Na+/K+-ATPase activity specifically limited to the cortical collecting duct in PAN rats. To further define whether this phenomenon was dependent on the chemical induction of the nephrotic syndrome or was a general phenomenon observed in glomerulonephritis, we measured Na+/K+-ATPase activity in nephron segments from mice with spontaneous lupus-like nephritis. Hydrolytic activity of Na+/K+-ATPase was measured in three isolated nephron segments: proximal convoluted tubule, thick ascending limb and cortical collecting duct. The Na+/K+-ATPase activities were measured in PAN rats, sham-injected controls, and in (MRL x BXSB) F1 male mice which develop a well established spontaneous lupus-like glomerulonephritis by 4 months of age and their controls. Control mice have the same genetic background, but lack the Yaa mutant gene responsible for autoimmune acceleration and are free of glomerular lesions at 4 months of age. In (MRL x BXSB) F1 male mice, Na+/K+-ATPase was similar to control mice in the proximal convoluted tubule and the thick ascending limb. In contrast, cortical collecting duct Na+/K+-ATPase activity was two times higher in (MRL x BXSB) F1 mice than controls. These results were identical to those observed in PAN rats compared to their sham-injected controls studied 7 days after an intraperitoneal injection of puromycin or isotonic saline, respectively. Enhancement of Na+/K+-ATPase activity localized to the cortical collecting duct is a general characteristic of glomerulonephritis independent of its mode of induction, i.e. chemical versus autoimmune. Therefore, the experimental model of PAN is suitable to study the underlying mechanisms leading to Na+/K+-ATPase dysfunction.

Inefficient processing impairs release of RNA from the site of transcription.

We describe here for the first time the site of retention within the nucleus of pre-mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human beta-globin genes that are either normal or defective in splicing or 3' end formation. Nuclear transcripts of both wild-type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, alpha-amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild-type human beta-globin RNA. In contrast, pre-mRNA mutants defective in either splicing or 3' end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3' end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.

Cerebellar learning in the vestibulo–ocular reflex

The vestibulo-ocular reflex, because of its close relationship with the cerebellum and its marked adaptiveness, has become a model system for studying the functions of the cerebellum. It has been hypothesized that an evolutionarily old part of the cerebellum, the flocculus, forms a modifiable accessory pathway for the vestibulo-ocular reflex arc for adaptive control, and that the modification is due to the synaptic plasticity induced by retinal errors conveyed by a unique structure of the cerebellum, the climbing fibers. The flocculus hypothesis has been supported by several lines of evidence, including lesioning or functionally impairing the flocculus and recording the activity of flocculus Purkinje cells, and, more recently, from pharmacologically or genetically inhibited synaptic plasticity, which produces long-term depression. There has also been debate on a possible site for memory retention in vestibulo-ocular-reflex adaptation, and about the signal content in flocculus Purkinje cells. This article reviews recent studies on the learning mechanisms of the cerebellum that underlie the adaptation of the vestibulo-ocular reflex.

Ethanol-induced effects on expression level, activity, and distribution of protein kinase C isoforms in rat liver Golgi apparatus

Acute ethanol administration induces significant modifications both in secretive and formative membranes of rat liver Golgi apparatus. The decrease in glycolipoprotein secretion and their retention into the hepatocyte contribute to the pathogenesis of alcohol-induced fatty liver. Molecular and cellular mechanisms behind the ethanol-induced injury of the liver secretory pathway are not yet completely defined. In this study on intact livers from ethanol-treated rats, the involvement of the Golgi compartment in the impairment of hepatic glycolipoprotein secretion has been correlated with changes in the expression level, subcellular distribution and enzymatic activity of protein kinase C (PKC) isoforms. Acute ethanol exposure determined a translocation of classic PKCs and δ isoform from the cytosol to cis and trans Golgi membranes, the site of glycolipoprotein retention in the hepatic cell. A marked stimulation of cytosolic ε PKC activity was observed throughout the period of treatment. The presence of activated PKC isozymes at the Golgi compartment of alcohol-treated rat livers may play a role in hepatic secretion and protein accumulation. Direct and indirect effects of ethanol consumption on PKC isozymes and Golgi function are discussed.

Enhancement of the leukocyte–endothelial cell interaction in collecting venules of skeletal muscle by protamine

Objective: A transient but severe systemic leukopenia regularly occurs after the antagonization of heparin by protamine in patients and in animals. The aim of the present study was to investigate the site and mechanisms of white blood cell retention during this transient leukopenia by studying the leukocyte–endothelial cell interaction in skeletal muscle venules. Methods: Syrian golden hamsters were equipped with a dorsal skinfold chamber for intravital fluorescence microscopy and arterial and venous catheters for drug infusion, blood pressure measurement, and blood sampling. Microhemodynamic parameters and leukocyte–endothelial cell interactions were observed in one single collecting venule per animal after intravenous infusion of saline solution (control, n = 10), of protamine ( n = 9), and after infusion of heparin followed by either intravenous protamine ( n = 9) or intraarterial protamine ( n = 9). Results: All parameters remained unchanged in the control group. Whereas venular diameters remained unchanged, protamine transiently increased arterial blood pressure and venular erythrocyte velocity in all groups. Systemic leukocyte counts and the venular leukocyte discharge concentration decreased concurrently after protamine administration by about 60% to 70% at 2 minutes while the fraction of rolling leukocytes and the number of adherent leukocytes remained unchanged. Two and one-half minutes later, systemic leukocyte counts and venular discharge concentrations normalized while the fraction of leukocytes rolling slowly along or adhering firmly to the venular endothelial wall increased considerably and similarly in all groups receiving protamine. Myeloperoxidase (an indicator of polymorphonuclear leukocytes) determination in 20 separate hamsters 2 minutes after protamine infusion revealed increased myeloperoxidase activity exclusively in the lungs. Conclusion: The response of leukocytes to protamine infusion with or without prior heparinization is biphasic: initial retention of leukocytes in the lungs is followed by enhanced leukocyte–endothelial cell interaction in the systemic circulation. (J Thorac Cardiovasc Surg 1997;113:784-91)

Ethanol-induced retention of nascent proteins in rat hepatocytes is accompanied by altered distribution of the small GTP-binding protein rab2.

Chronic ethanol consumption induces hepatocellular retention of nascent proteins leading to hepatomegaly. While the molecular mechanisms behind this impairment are undefined, it has been predicted that protein retention results from a disruption of vesicle-mediated secretory processes. Small GTP-binding proteins (rab proteins) have recently been implicated in the regulation of vesicular trafficking in eukaryotic cells. Our objectives were to identify intracellular sites of ethanol-induced protein retention and to determine whether the distribution of secretory rab proteins was altered by ethanol. Transport of hepatic proteins along the secretory pathway in livers from control and ethanol-fed rats was analyzed using subcellular fractionation and immunoprecipitation in the context of in vivo pulse-chase experiments. We show that pre-Golgi and Golgi compartments, as well as secretory vesicles, are sites of ethanol-induced retention of nascent soluble and transmembrane secretory proteins. These results are supported by immunofluorescence localization of hepatic proteins on liver sections. Further, immunoblot analyses of hepatic subcellular fractions from ethanol-damaged livers indicate a dramatic reduction in the association of rab2 with a Golgi compartment as compared with controls. In contrast, rab6 and alpha-mannosidase II, Golgi marker proteins, appear unchanged. These studies provide a detailed analysis of the intracellular site of ethanol-induced protein retention in the hepatocyte and lend novel insight into a potential mechanism behind this impairment. The effects of ethanol exposure on rab proteins and Golgi function are discussed.

Increased intestinal amino-acid retention from the addition of carbohydrates to a meal

Carbohydrates, added to a protein meal, are known to enhance the efficiency ofdietary protein utilisation. However, the respective roles of the gut and liver in relation to this enhanced efficiency are not known. Therefore, we studied amino-acid, ammonia, urea, glucose and lactate fluxes for 6 h across the portal drained viscera and liver in conscious, multicatheterised pigs of approximately 25 kg body weight after receiving a protein meal with added carbohydrates, a pure protein meal or a control meal. Additional carbohydrate caused a net glucose efflux in the portal drained viscera andincreased arterial blood insulin levels. The appearance of amino-acids in the portal blood declined by some 30%, in spite of the dietary true amino-acid digestibility being approximately 95%. Liver uptake of most amino-acids was lower and there was a lower liver urea production. Finally, there was a smaller postprandial increase in the arterial blood concentration for most of the amino-acids. The results of this study suggest that inclusion of maltodextrin in the diet increases the net retention of meal-derived amino-acids in the portal drained viscera. The lower urea production and liver amino-acid uptake suggest a lower nitrogen loss. The gut could be an important site for nitrogen retention induced by the addition of carbohydrates to a protein meal.

Particle retention during respiration and particulate feeding in the suspension-feeding blackfish,Orthodon microlepidotus

Juvenile blackfish (Orthodon microlepidotus) retained styrene microspheres (30–90 μm) during respiration in the absence of food. The fish retained significantly more microspheres when particulate feeding on Artemia adults, suspension feeding on Artemia nauplii, or in the presence of Artemia extract than when respiring in the absence of food. The frequency distribution of microsphere sizes retained during respiration did not differ significantly from that retained during feeding, a result that is consistent with the hypothesis that the site and method of particle retention during respiration and particulate feeding do not differ from those operating during suspension feeding. There was significant interindividual variability in the number of particles retained. Some individuals retained consistently fewer microspheres than other individuals of similar size and age, suggesting that further study is needed to quantify interindividual variation in other factors (e.g., stroke volume, ventilation rate, or mucus secretion) that have the potential to affect particle retention rates. Our results have implications for the energetics of suspension-feeding fishes, the impacts of suspension-feeding fishes on plankton communities, and the exposure of these fishes to toxic phytoplankton strains and particle-bound organic chemicals.

Effect of blood flow on the leukocyte-endothelium interaction in pulmonary microvessels.

Circulating leukocytes are retained in the microcirculation of the lung. The site of leukocyte retention, however, is still a subject of controversy, and the effects of microvascular blood flow on the leukocyte-endothelium interaction in pulmonary microvessels are unknown. We used in vivo fluorescence microscopy to analyze microhemodynamics and the flow behavior of in vivo-labeled leukocytes in pulmonary arterioles, venules, and alveolar capillaries. Microvascular blood flow was altered by variation of cardiac output. Leukocytes were found to roll and to stick on arteriolar and more pronouncedly on venular endothelium. During their passage through alveolar capillaries, a fraction of passing leukocytes became static for 0.1 to > 5 s. Under control conditions, leukocytes were concentrated approximately 8-fold more in arterioles and 24-fold more in venules than in the blood passing through these vessels. The concentration in capillaries was 1.5 times greater than in venules. The velocity of rolling leukocytes in arterioles and venules correlated significantly with the shear rate in these vessels, whereas the density of sticking cells was negatively correlated with the shear rate. The differences between leukocyte rolling and sticking in arterioles and in venules cannot be explained by respective hemodynamic conditions. In alveolar capillaries, the percentage of temporarily static leukocytes and the time of their stasis were inversely correlated with red-blood-cell (RBC) velocity. We conclude that leukocytes are retained in pulmonary arterioles, venules, and alveolar capillaries according to microvascular blood flow and endothelial factors.

Transmission of Viruses by Plant Nematodes

ABSTRACf Longidorid and trichodorid nematodes transmit nepo- and tobraviruses, respec­ tively. A specific association exists between vectors and their viruses which is a consequence of the nature, site and mechanism of virus particle retention within the vecto r. It is correlated with the serological properties of the virus coat protein and detennined by the RNA-2 segment of the virus genome and by an inherited character of the vector. The virus coat protein is probably involved in the recognition process between vector and virus but is not the sole determinant for transmission of tobraviruses. Genetic changes made to proteins present in the RNA-2 segment of pea early.browning tobravirus have been used to reveal the probable involvement of several proteins in vector transmission.